The glutathione S-transferases catalyze the nucleophilic addition of the sulfur of glutathione (GSH) to a wide variety of endogenous and xenobiotic substrates hearing electrophilic functional groups. They are arguably the single most important enzyme in the metabolism and detoxification of alkylating agents including cancer chemotherapeutic drugs and metabolites of environmental pollutants. The catalytic versatility of this group of enzymes derives from a large number of isoenzymes with broad, overlapping substrate preferences. It is the thesis of this proposal that a thorough understanding of the participation of this group of enzymes in the metabolism of drugs and xenobiotics must be based on a detailed knowledge of the relationship between molecular structure and catalysis. The research plan integrates the disciplines of physical organic chemistry, enzymology and structural biology in an attempt to elucidate the details of substrate recognition and catalysis by the GSH transferases. The investigations will focus on isoenzymes from the mu and sigma classes. Mechanistic investigations are proposed to (i) explore the influence of the electrostatic environment of the sulfur in the E-GSH complex by site-general and site-specific incorporation of unnatural amino acids; (ii) define the internal equilibrium and stereochemistry with reversible Michael-acceptor substrates; and (iii) elucidate the structural basis for the apparent cooperativity between the subunits in the dimeric enzyme by rapid kinetic, equilibrium and mutagenesis techniques. Construction and characterization of new enzymes with altered catalytic properties will be attempted in order to elucidate the role of specific structural elements in the catalytic mechanism and substrate recognition. These constructs will include; (i) deletion mutants encoding enzymes lacking the mu loop and C-terminal tail to assess the role of these structural elements in catalysis and product release; (ii) domain interchange (hybrid) constructs of class mu isoenzymes to elucidate the global effect of domain 11 on catalytic specificity; and (iii) deletion mutants lacking all or part of domain II to determine the necessity of the domain for catalytic activity and protein stability. Structural investigations will be pursued in several areas including; (i) crystallization and structure determination of the 3-4 heterodimer of the class mu isoenzyme from rat; (ii) structure determination of isoenzyme 4-4 at high resolution; (iii) crystallization and structure determination of the lens S-crystallins of cephalopods related to the class sigma GSH transferase; (iv) crystallization and structure determination of the tetradeca-(3- fluorotyrosyl)-isoenzyme 3-3; (v) crystallization and structure determination of site-specific mutants of mechanistic and structural interest and; (vi) crystallization and structure determination of selected structurally modified or truncated enzymes. The knowledge derived from all of these studies will ultimately be used as a basis for the design of proteins with new catalytic and physical properties.